RESUMO
Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1ß. We previously showed that IL-1ß release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1ß release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1ß release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1ß release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1ß release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1ß, but resulted in the retention of mature IL-1ß within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1ß was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1ß release from viable cells. This study expands on the molecular pathways that promote IL-1ß in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1ß release during infection.
Assuntos
Caspase 8 , Interleucina-1beta , Toxoplasma , Toxoplasmose , Humanos , Caspase 1/metabolismo , Caspase 8/metabolismo , Gasderminas , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Monócitos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Toxoplasmose/metabolismoRESUMO
IL-1ß is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1ß; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1ß via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1ß release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1ß transcripts and the production of pro-IL-1ß. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1ß production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1ß protein levels and IL-1ß release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1ß transcriptional activation. IL-1ß release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1ß to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1ß in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Quinase Syk/metabolismo , Toxoplasmose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Células Cultivadas , Humanos , Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Monócitos/imunologia , Monócitos/microbiologia , NF-kappa B/genética , Proteínas de Ligação a Fosfato/genética , Transdução de Sinais , Quinase Syk/genética , Toxoplasma/fisiologia , Toxoplasmose/imunologia , Toxoplasmose/microbiologiaRESUMO
Intracellular pathogens strike a delicate balance between maintaining their survival within infected cells, while also activating host defense mechanisms. Toxoplasma gondii is a protozoan parasite that initiates a variety of host signaling pathways as it invades host cells and establishes residence in a parasitophorous vacuole. Recent work has highlighted the interplay between T. gondii infection and innate immune pathways that lead to inflammation, several of which converge on caspases. This family of cysteine proteases function at the crossroads of inflammation and cell death and serve as a key target for parasite manipulation. This review focuses on the interaction of T. gondii with caspase-dependent inflammatory and cell death pathways and the role of parasite effector proteins in modulating these processes.
Assuntos
Capparis , Toxoplasma , Humanos , Toxoplasma/fisiologia , Capparis/metabolismo , Caspases/metabolismo , Transdução de Sinais , Inflamação , Proteínas de Protozoários/metabolismoRESUMO
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression.